Plexins are single-pass transmembrane receptors that serve as the primary receptors for the guidance molecules semaphorins. Some semaphorins also require the neuropilin co-receptor for binding and activating plexin. Semaphorin/plexin/neuropilin-mediated signaling is essential for many processes, including the development of the nervous system and the cardiovascular system. Malfunction of this signal pathway is associated with diseases such as neurological disorder and cancer. A better understanding of the mechanisms of this pathway will provide a foundation for developing targeted therapies for these diseases and improving neuronal regeneration after injury. Plexin and neuropilin are both large proteins, and use their N-terminal membrane distal domains to bind semaphorin. Semaphorin are dimeric molecules, and activate plexin by inducing the formation of the active dimer. One major remaining mechanistic question is how the membrane proximal and transmembrane regions in plexin and neuropilin couple the semaphorin binding at the N-terminal domains to the activation of the plexin cytoplasmic domain, which relays the signal to downstream pathways. There is evidence suggesting that the membrane proximal and transmembrane regions play active roles in plexin activation. Another outstanding question concerns how many of the newly identified binding partners of plexin contribute to the signaling. The proposed research will be focused on addressing these mechanistic questions. In Aim 1, we will analyze how the interactions mediated by the transmembrane region of plexin regulate the formation of the plexin active dimer. We will design plexin constructs that contain the transmembrane region and cytoplasmic region but not the N-terminal autoinhibitory domains. These constructs are expected to form the active dimer spontaneously in the absence of semaphorin binding. We will determine the structure of this active dimer through either X-ray crystallography or cryo-electron microscopy to visualize how the transmembrane region interacts and promotes the formation of the plexin active dimer. In Aim 2, we will pursue the structure of full-length plexin and neuropilin in complex with semaphorin by using cryo-electron microscopy. These structures will provide a direct view of the entire receptor/ligand complex, and reveal how the membrane proximal and transmembrane regions couple the binding of semaphorin at the N-terminal domains to the cytoplasmic domains. In Aim 3, we will investigate the interactions and regulation of plexin by regulatory proteins. Some of these proteins may only exert their regulatory function in the context of the lipid bilayer, which we will investigate by using plexin reconstituted in lipid discs. The structural studies will be correlated by in vitro biophysical and cell-based functional assays. In addition, new approaches developed for the plexin system will be used to study other transmembrane signaling proteins in order to gain a general understanding of the principles of transmembrane signaling.